Lipidic furans and retinol palmitate compositions useful for skin therapeutics

ABSTRACT

A composition of matter useful for enhancing the metabolism of the skin and mucosal tissue and thereby improving its health and appearance. The composition of matter includes a lipidic furan and retinol palmitate. The lipidic furan has a structure: furan--R, wherein R is a straight C 9-36  alkyl chain, having the formula CH 3  --(CH 2 ) n  --(CH 2 ) n  CH 3 , (n=8 to 35). R can also be a straight unsaturated C 9-36  alkenyl chain with a single double bond, of the formula --(CH 2 ) n  --CH═CH--(CH 2 ) m  --CH 3 , wherein n and m≠0 and n+m=6 to 33. R can be a straight unsaturated C 9-36  chain, containing two to six double bonds, with the formula CH 3  --(CH 2 ) m  --(CH═CH) x  --(CH 2 ) n  --, wherein m, x and n≠0, m+2x+n=8 to 35. R can likewise be a straight unsaturated C 9-36  chain containing one to six double bonds and one triple bond, having the formula CH 3  --(CH 2 ) m  --C.tbd.C--(CH═CH--CH 2 ) x  --, wherein m and x≠0 and m+3x=6 to 33. R may be a straight unsaturated chain C 9-36  containing one to six double bonds and two to six triple bonds, having the structure CH 3  --(CH 2 ) m  --(C.tbd.C--CH 2 ) y  --(CH═CH--CH 2 ) x  --(CH 2 ) n  --, wherein m, y, x and n≠0 and m+3y+3x+n=8 to 35.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of lipidic furans, and moreparticularly to the use of certain lipidic furans characterized by analiphatic hydrocarbon-chain with a minimum of nine carbon atoms,attached to the number two position on a furan ring, in combination withretinol palmitate, for improving the condition of human skin.

2. Description of Prior Art

For many years, there has been much folklore surrounding the beneficialqualities of avocados and avocado oil in improving the skin.

It has been observed that workers handling avocado oil havesignificantly moisturized, smoother appearing hands. It has also beenobserved that a group of Central American Indians, whose diet included ahigh proportion of fresh avocados and avocados in a slightly warmeddecoction, had significantly healthier looking skin than tribes innearby areas that did not include avocado in their diets in significantproportions.

Some skin care specialists recommend placing packs of freshly mashedavocado pulp to the skin, as well as various preparations containingavocado and avocado oils to freshen and revitalize the skin and improveits appearance.

Over the years, there have been attempts to determine exactly what it isin the avocado which improves the appearance of the skin. However, theseefforts have been unsuccessful.

In U.S. Pat. No. 4,386,067 to Guillon, the non-saponifiable fraction ofavocado oil was mixed with the non-saponifiable fraction of soya-beanoil to form a mixture which aids in moisturizing the upper layer ofskin. However, Guillon states that the composition of thenon-saponifiable fraction is complex and is not completely known. Noreference to lipidic furans is made.

U.S. Pat. No. 4,324,802 to Koulbanis et al. discloses that thenon-saponifiable fractions of avocado oil and/or soy bean oil haveuseful dermatological properties for use in cosmetic compositions toimprove the appearance of aging, dry or wrinkled skin. Again, however,the composition of the non-saponifiable fractions of the avocado oil isnot identified.

In the journal article "New Compounds From Avocado Pear", by Y.Kashman, 1. Neeman and A. Liftshitz, Tetrahedron Vol. 25, pp. 4617-4631,compounds of Structure I, II and III with molecular weights 246, 248 and250 were disclosed. No mention was made of the use of these compounds.

In the journal article "Partial Isolation Of and Characterization Of ANew Natural Inhibitor Of Lysyl Oxidase From Avocado Seed Oil", by Mr. J.Werman, S. Mokady, and I. Neeman, J. Agric. Food Chem. 1990, 38,2164-2168, it was reported that a unsaponifiable component ("C") ofavocado seed oil inhibits the action of lysyl oxidase. Lysyl oxidaseinitiates cross-links in collagen and in collagenic connective tissue,including skin, and an over abundance of these crosslinks results inwrinkles, and a lack of elasticity in the skin. Werman et al. notedcomponent "C" was derivable only from the unsaponifiable fraction ofunrefined avocado oil or avocado seed oil. The researchers were unableto determine the composition of component "C", which was stated as being"probably a mixture of some polyalcoholic compounds, one of them havinga molecular weight of 248 and being composed of a 17-carbon aliphaticchain with a furan ring." p. 2167

In "The Avocatins--A New Class of Natural Products" by H. MagalhaesAlves, et al., An. Acad. brasil, Cieno, (1970), 42 (suplemento),compounds referred to as avocatins were discussed. These compounds areextractable from seeds of avocado pears, and have structures includingthe following alkyl furans:

furan (2)--(CH₂)--CH═CH₂, furan (2)--(CH₂)₁₁ C.tbd.CH

furan (2)--CH═CH--(CH₂)₉ --CH═CH₂,

furan (2)--CH₂ --CH═CH--(CH₂)₈ CH═CH₂

Each of these alkyl furans of the Alves, et al., article have a 13carbon alkyl chain having one or two double bonds, or a single triplebond. The chain is attached to the number two position of a furan ring.

The short communication entitled "Isolation And Structure Elucidation ofGrowth Inhibitors For Silkworm Larvae From Avocado Leaves" by Ching-FunChang, et al., Agr. Biol. Chem., 39 (5), 1167˜1168, 1975 discloses agrowth inhibitor for Silkworm larvae, C₂₃ H_(C40) O₄ which when treatedwith p-toluene sulfonic acid, yielded a new compound C₂₁ H₃₄ O,MW302,which is a two-substituted furan. The Chang et al. article fails todisclose the exact structure of this molecule or any cosmetic ortherapeutic use of the compound for human skin use.

The journal article "A New Lipid Component Identified in Avocado Pear byGC-MS and NMR Spectroscopy", by N. Frega, et al. This journal articlediscloses the discovery of a new cyclic lipic compounds in the extractsof the unsaponifiable extracts of avocado having the followingstructure: ##STR1##

Not only is this compound not a furanyl lipid, but there was nodiscussion of for what these compounds might be useful.

Lastly, the article "Fatty Acids, Part XVI: The Synthesis Of AllIsomeric C₁₈ Furan-Containing Fatty Acids", by M. S. F. Lie Ken Jie andC. H. Lam, Chemistry and Physics of Lipids 21 (1978) 275-287, discloses,as an intermediate in the synthesis of two and five substituted furaniccompounds, the following structure:

    CH═CH(CH.sub.2).sub.y X,

X=CH₃ or COOCH₃

y=number of methylene groups

However, there is no discussion of what these compounds might be usefulfor.

Although a substantial amount of effort has been directed to elucidatewhat it is in avocados which make avocados good for the skin, theseefforts have fallen short. No one in the past has identified thespecific compounds from avocado which have useful cosmetic andtherapeutic effects.

SUMMARY OF THE INVENTION

The invention is directed to a series of specific chemical compounds,termed lipidic furans, and their specificity to types I and IIIcollagen, both of which are present in large amounts in the skin andmucosal tissues, (e.g. the tissue in the gums), and the use of theselipidic furans, in combination with retinol palmitate, for enhancing themetabolic activities in these tissues. The enhanced metabolic activityexhibits itself by causing increases in the elasticity, protein content,DNA content, and for skin, increases in thickness of the epidermas anddermas. The enhanced metabolic activity is further exhibited byincreases in the fibroblast population, total protein synthesis,collagen synthesis, heightened glucose metabolism in the treatedtissues, and an overall improvement in the appearance and feel of theskin and mucosal tissue. These compounds are useful to combat dermalatrophy by slowing, and in many cases reversing the thinning skin, fineand coarse wrinkles, microvascular loss, capillary breakage anddeformity brought about by chronological and photo aging of the skin. Insum, these compounds have the ability to markedly enhance the metabolismof skin and mucosal tissues.

The lipid furan portion of the composition of the invention has analiphatic hydrocarbon with a minimum of nine carbon atoms attached tothe number two position of the furan ring. These compounds have thegeneral formula: ##STR2## wherein R is a straight saturated chain alkyl--(CH₂)_(n) CH₃, and n=8 to 35. R may also be a straight unsaturatedalkyl chain C₉₋₃₆ with a single double bond, of the formula --(CH₂)_(n)--CH═CH--(CH₂)_(m) --CH₃, n and m≠0 and n+m=7 to 34. R may also be astraight unsaturated C₉₋₃₆ chain, containing two to six double bonds,with the formula CH₃ --(CH₂)_(m) --(CH═CH)_(x) --(CH₂)_(n) --, whereinm, x and n≠0, m+2x+n=8 to 35. R may also comprise a straight unsaturatedC₉₋₃₆ chain containing one to six double bonds and one triple bond,having the formula CH₃ --(CH₂)_(m) --C.tbd.C--(CH═CH--CH₂)_(x)--(CH₂)_(n) --, wherein m, x and n≠0 and m+3x+n=6 to 36, or R maycomprise a straight unsaturated chain C₉₋₃₆ containing one to six doublebonds and two to six triple bonds, having the structure CH₃ --(CH₂)_(m)--(C.tbd.C--CH₂)_(y) --(CH═CH--CH₂)_(x) --(CH₂)_(n) --, wherein m, y, xand n≠0 and m+3y+3x+n=8 to 35.

Illustrative examples of lipidic furan compounds falling under thedefinition include the following:

Capric furan (2 Furanyl nonane)

Lauric furan (2 Furanyl hendecane)

Myristic furan (2 Furanyl tridecane)

Margaric furan (2 Furanyl hexadecane)

Arachidic furan (2 Furanyl nonadecane)

Lignoceric furan (2 Furanyl docosane)

Lauroleic furan (11-(2 Furanyl)-8-cis-hendecene)

Palmitoleic furan (15-(2 furanyl)-8-cis-pentadecene)

Cis-vaccenic furan (17-(2 furanyl)-10-cis-heptadecene)

Erucic furan (21-(2 furanyl)-12-cis-heneicosene)

Nervonic furan (23-(2 furanyl)-14-cis-trieicosene)

Linoleic furan (17-(2 furanyl)-8-11-cis-cis-heptadecadiene)

Arachidonic furan (19-(2furanyl)-4-7-10-13-cis-cis-cis-cis-nonatetraene)

Crepenynic furan (18-(2 furanyl)-octadec-cis-8-en-11-yl)

Biological Activity

The lipic furan components of the invention enhance the metabolism ofthe dermis of humans and animals and have therapeutic value when appliedtopically in a proper cosmetic carrier vehicle, or taken orally. Forexample, the carrier vehicle may comprise an oil-water emulsion, awater-oil emulsion, or micro emulsion or micro encapsulated skinmoisturizing formulation.

The Applicants have found that while all of the compounds listed aboveare useful, in particular the compound pictured below, 17-(2Furanyl-8-11-cis-cis-heptadecadiene ("Linoleic Furan"), in combination¹with retinol palmitate, has profound and beneficial effects on theepidermis and dermis of the skins and significantly the moisturizationand smoothness of the skin.

The Applicants have found that several beneficial therapeutic effectsare caused by the use of Linoleic Furan in combination with retinolpalmitate as follows:

a) An increase in the number of cells in the skin as determined by DNAcontent and changes to the skin cells.

b) An increase in dermal and epidermal tissue as determined by proteincontent.

c) An increase in dermal metabolism as determined by ¹⁴ C!-glucosemetabolism.

d) An increase in protein and collagen synthesis as determined by ³H!-proline incorporation into protein and collagenase digestibleprotein.

e) An increase in dermal collagen content as determined by an increasein hydroxyproline.

f) An increase in the thickness of both the dermis and epidermis of skintreated with the Linoleic Furan in a suitable cosmetic formulation.

The above changes indicate that skin metabolism is "activated" bytopical application of Linoleic Furan.

Empirical Experiments to Prove Structure of Linoleic Furan

Linoleic Furan can be extracted from the seed or pit of avocados, or canbe synthesized. Linoleic Furan, which is either obtained by extractionfrom avocados, or produced synthetically, has a molecular weight of 302Daltons, is oily, and has a light yellow color.

Linoleic Furan was obtained from the avocado oil by separation. Theseparation scheme involved normal phase liquid chromatography on highlyactivated silica gel using hexane as the mobile phase. In thisseparation, 10 fractions were collected. Polarity was increased untilthe final fraction was removed by the use of 100% methanol. Theseparated fractions were subjected to mass spectroscopy. The majorcomponent of each fraction was determined by these analyses. Below arelisted the results of these analyses. In addition to the materialslisted in Table 1, 42 mg of material were insoluble in the originalhexane solvent when 20.5 g of starting avocado seed extract were used.

                  TABLE 1    ______________________________________                                   %             MATERIAL              COM-    FRACTION (weight/weight)       POSITION    ______________________________________    A        Alkanes C.sub.27 H.sub.56, C.sub.29 H.sub.60, C.sub.31 H.sub.64                                   6.1    B-F      Alkenyl furans of the type                                   62.0     ##STR4##                     ##STR5##    "B" = MW = 276  "C" = MW = 278     ##STR6##    "D" = W = 302    G        A mixture of F & H    2.3    H        Tocopherol            8.3    I        Sitosterol            13.6    J        a tar like substance  6.7    ______________________________________

Upon testing these various fractions, it was found that Fraction Dappeared to contain the most active material in terms of promotingdermal thickening (Table 2). This particular alkenyl furan containingfraction contained within it an alkenyl furan with a 302 Daltonsmolecular weight.

                  TABLE 2    ______________________________________    EFFECT OF THE TOPICAL APPLICATION    OF VARIOUS FRACTIONS OF    AVOCADO SEED EXTRACT ON SKIN WEIGHT                   PUNCH WEIGHT    TREATMENT GROUP                   (mg wet weight/6 mm punch biopsy)    ______________________________________    Control        11.9 ± 0.1    Control Vehicle                   11.7 ± 0.2    Fraction A     11.3 ± 0.4    Fraction D        13.0 ± 0.4*,**    Fraction F     11.8 ± 0.3    Fraction H     11.0 ± 0.3    Fraction J     12.3 ± 0.4    ______________________________________     Values represent the mean ± S.E.M. of 5 animals (S.E.M. = Standard     Error of the Mean)     *Indicates significantly different from control at p < 0.05     **Indicates significantly different from vehicle control at p < 0.05.

In order to obtain more purified material with which subsequent workcould be done, normal phase HPLC was initiated to separate Fraction Dfrom the pit extract using preparative normal phase silica columns. Withthis system, a separation which gave three distinct peaks when monitoredat 280 nm was achieved. In addition, thin layer plate chromatographyalso demonstrated that there was a separation of three distinctmoieties. The three fractions which were obtained are: (1) aparaffin-like material, (2) a light yellow oil and (3) a dark yellowoil. The recovery of this material ranges from 40-55% of the appliedmaterial. The second step in the isolation is normal phase HPLC of thelight yellow oil. A sufficient quantity of these three fractions wastaken for additional purification. Using reverse phase HPLC on a C-18column with an acetonitrile/tetrahydrofuran mobile phase, it waspossible to divide the materials into families based on UV absorbance(Table 3).

Mass spectroscopy demonstrated that a separation of the 302 MW compoundwas achieved and that it eluted in the third fraction.

                  TABLE 3    ______________________________________    SOURCE OF  FRACTIONS    MATERIAL   OBTAINED    COMMENTS    ______________________________________    Normal Phase    HPLC Fractions    #28-30     A           max, absorbance at 266 nm               B           colorless material which is                           greatest quantity (>80%)                           max. absorbance is <210 nm                           MW-302    #9-10      C           max. absorbance is at 266 nm               D           max. absorbance is at 216 nm    #16        E & J       max. absorbance is at 266 nm               F, G, H & I max. absorbance is at 216 nm    ______________________________________

Following these separations, it was found that the material B obtainedby reverse phase chromatography accounted for better than 80% of thematerial in the fraction #28-30 from normal phase HPLC (Table 3). Thus,it appeared that this material was in the "dermal active material" andwas the most prevalent molecular species in the dermal active material.

Although this work indicated that the alkenyl furan (MW 302) of fractionD of Table 2 was the molecule of interest, the precise structure of thiscompound was unknown. From the mass spectroscopy data and from the NMRdata, it was evident that the structure contained two double bonds.Ozonolysis of the isolated compound was performed and mass spectroscopyand gas chromatography/mass spectroscopy on the oxidized fragments wascarried out. This gave the structure D as presented in Table 1. However,this did not determine if the double bonds were "cis" or "trans".

To determine the absolute structure of the molecule, the ¹ H NMRspectrum in addition to the ¹³ C NMR spectrum of the compound wasexamined. The ¹ H NMR spectrum in CDCl₃ is identical to the spectrum oflinoleic acid (an all cis compound) for the common chemical groups withthe exception of the α methylene to the carboxyl and the furan. Strongerevidence for a common double bond stereochemistry comes from the ¹³ CNMR spectra. Trans double bonds would cause shifts of the α carbons upto 5 ppm in comparison to cis double bonds. The α carbons were assignedby heteronuclear correlation or decoupling and compared. The shifts wereless than 0.1 ppm different which indicates that the double bonds havethe same stereochemistry as in linoleic acid.

In addition, Raman spectroscopy on the purified 302 compound wasperformed. This study demonstrated a 1660 cm⁻¹ peak which correspondswell to what would be expected with a cis, cis configuration. From this,it was determined that the active fraction "D" is indeed Linoleic Furan.

Empirical Evidence of Biological Activity of Linoleic Furan

Experiments were conducted to demonstrate that Linoleic Furan increasesskin protein synthesis and thereby increases skin tissue as determine bydermal DNA and protein content.

Four sets of 8 female hairless mice were treated each day for 14 dayswith 0.1 ml of each formulation. The formulations were as follows:

No treatment

Control Vehicle--oil-in-water emulsion

Vehicle with 3% (w/w)* natural isolated Linoleic Furan

Vehicle with 3% (w/w) synthetic Linoleic Furan

*=weight/weight

In this and all subsequent studies, Linoleic Furan was formulated in anoil-in-water emulsion. The oil phase of this emulsion was 16% (w/w), thewater phase ranged from 81-84% (w/w) and the concentration of LinoleicFuran ranged from 0-3% (w/w).

Skin composition and morphometric measurement were made as described inThe Effect of Retinyl Palmitate on Skin Composition and Morphometry, byCounts, D., Skreko, F., McBee, J. and Wich; Am. J. Soc. Cosmet. Chem.(1988) 39:235-240).

After treatment, the animals were sacrificed by cervical dislocation,the skin was removed and the subcutaneous tissue was dissected from theskin. A 6 mm punch biopsy was used to obtain a biopsy from the skin foranalyses. For protein synthesis the biopsy was incubated in mediacontaining 2,3-³ H! proline. Following 4 hours of incubation, the tissuewas placed in 10% weight/volume (w/v) trichloroacetic acid and analyzedfor incorporation of 2,3-³ H! proline into protein and collagenasedigestible protein (Counts, D., Knighten, P. and Hegreberg, G.,Biochemical Changes in the Skin of Mink with Ehlers-Danlos Syndrome:Increased Collagen Biosynthesis in the Dermis of Affected Mink. J.Invest. Dermatol. (1977) 69:521-526).

RESULTS

The analyses of the skin composition demonstrated that the addition of3% of either natural or synthetic Linoleic Furan had the same effect onskin composition and weight (Table 4). Although all of the alkenyl furancontaining formulations significantly increased protein synthesis, notall significantly increased collagen synthesis in this study (Table 5).Therefore, it is possible to use synthetically produced Linoleic Furanrather than isolated natural Linoleic Furan for skin treatmentformulations.

The effect of Linoleic Furan on skin composition was confirmed by thechange in skin morphometry as determined by microscopy (Table 6). Thus,topical application of Linoleic Furan causes an increase in skinthickness. These data suggest that treatment of skin on aging humanswith Linoleic Furan may reverse the dermal atrophy known to beassociated with human skin chronological and photo aging.

                                      TABLE 4    __________________________________________________________________________    EFFECT OF NATURAL AND SYNTHETIC LINOLEIC FURAN    ON THE SKIN OF HAIRLESS MICE            PUNCH   PROTEIN DNA     COLLAGEN    TREATMENT            WEIGHT  CONTENT CONTENT CONTENT    GROUP   (mg/punch)                    (mg/punch)                            (μg/punch)                                    (nmole HYP/Punch)    __________________________________________________________________________    Control 12.2 ± 0.7                    2.66 ± 0.19                            51.0 ± 7.8                                    109 ± 29    (no treatment)            (8)     (8)     (8)     (8)    Control Vehicle            13.6 ± 0.4                    3.11 ± 0.25                            57.0 ± 2.7                                    192 ± 28            (8)     (8)     (8)     (8)    3% Natural            18.1 ± 0.4*,**                    4.52 ± 0.31*,**                            96.7 ± 9.9*,**                                     185 ± 18*    Linoleic Furan            (8)     (8)     (8)     (8)    3% Synthetic            17.6 ± 0.4*,**                    4.07 ± 0.3*,**                            79.5 ± 3.7*,**                                    187 ± 26    Linoleic Furan            (8)     (8)     (8)     (8)    __________________________________________________________________________     Values represent the mean ± S.E.M. of the number of animals in     parentheses.     *Indicates significantly different from untreated control at p < 0.05.     **Indicates significantly different from the control vehicle treated grou     at p < 0.05.

                  TABLE 5    ______________________________________    EFFECT OF NATURAL AND SYNTHETIC LINOLEIC    FURAN ON SKIN PROTEIN AND COLLAGEN    SYNTHESIS IN THE SKIN OF HAIRLESS MICE                 TOTAL PROTEIN                             COLLAGEN                 SYNTHESIS   SYNTHESIS    TREATMENT GROUP                   (DPM  .sup.3 H!-PRO/Punch)    ______________________________________    Control        3836 ± 597 1895 ± 281    (no treatment) (8)           (8)    Control vehicle                   4145 ± 677 1892 ± 351                   (8)           (8)    3% Natural      6962 ± 1073*,**                                 2362 ± 517    Linoleic Furan (7)           (7)    3% Synthetic   6693 ± 790*,**                                 2561 ± 370    Linoleic Furan (6)           (6)    ______________________________________     Values represent the mean ± S.E.M. of the number of animals in     parentheses.     *Indicates significantly different from untreated control at p < 0.05.     **Indicates significantly different from the control vehicle treated grou     at p < 0.05.

                  TABLE 6    ______________________________________    EFFECT OF LINOLEIC FURAN OR    SKIN MORPHOMETRY OF HAIRLESS MICE                                      TOTAL              EPIDERMAL   DERMAL      THICK-    TREATMENT THICKNESS   THICKNESS   NESS    GROUP     (μm)    ______________________________________    Control   2.09 ± 0.07                          16.6 ± 1.3                                      33.7 ± 1.5    (no treatment)              (10)        (10)        (10)    Control vehicle              5.69 ± 0.27*                          22.1 ± 3.0                                      39.5 ± 3.4              (10)        (10)        (10)    3% Natural              10.1 ± 0.55*,**                          25.2 ± 1.7*,***                                      46.1 ± 1.9*    Linoleic Furan               (9)         (9)         (9)    ______________________________________     Values represent the mean ± S.E.M. of the number of animals in     parentheses.     *Indicates significantly different from untreated control at p < 0.05.     **Indicates significantly different from the control vehicle treated grou     at p < 0.05.

The Effect of Long Term Treatment With Linoleic Furan on Skin Metabolismand Morphometry

PURPOSE

To determine if the application of Linoleic Furan for extended periodsof time gives the same results as the two weeks of treatment.

PROTOCOL

Female hairless mice were treated with 0.1 ml of either Control vehicleor 3% Linoleic Furan incorporated into Control vehicle each day for 6weeks. At termination, the animals were biopsied for histology and skinbiochemical analyses as described above.

RESULTS

As can be seen in Table 7, six weeks of treatment dramatically increasedpunch weight, protein content and DNA content in the skin. The increasein protein synthesis observed after 14 days of topical applicationLinoleic Furan was sustained after 6 weeks of application. In this studycollagen synthesis was also increased (Table 8). The increase inepidermal, dermal and total skin thickness is also dramaticallysustained (Table 9). Finally, while six weeks of treatment with Controlvehicle alone increases (the number of fibroblasts within the dermis(Table 10), the addition of 3% Linoleic Furan to Control vehiclesignificantly increases the number of fibroblasts in the dermis over andabove the increase due to Control vehicle alone. Although treatment withControl vehicle also causes changes in all these parameters at 6 weeks,the effect is not as dramatic as the changes brought about by Controlvehicle plus 3% Linoleic Furan. These increases in tissue can bepartially explained by the increase in protein synthesis and the numberof dermal cells (fibroblasts) in the skin of the treated groups (Tables8 and 10).

                                      TABLE 7    __________________________________________________________________________    EFFECT OF LONG TERM TREATMENT (6 WEEKS)    WITH LINOLEIC FURAN ON SKIN COMPOSITION OF HAIRLESS MICE            PUNCH   PROTEIN DNA     COLLAGEN    TREATMENT            WEIGHT  CONTENT CONTENT CONTENT    GROUP   (mg/punch)                    (mg/punch)                            (μg/punch)                                    (nmole HYP/Punch)    __________________________________________________________________________    Control 10.9 ± 0.4                    0.94 ± 0.04                            40.4 ± 7.4                                    211 ± 31    (no treatment)            (10)    (10)    (10)    (10)    Control 13.6 ± 0.5*                    1.40 ± 0.16*                            47.1 ± 2.1*                                    202 ± 16    Vehicle  (9)     (9)     (9)     (9)    3% Natural            17.1 ± 0.4*,**                    1.90 ± 0.05*,**                            64.5 ± 2.7*,**                                    226 ± 15    Linoleic Furan            (10)    (10)    (10)    (10)    __________________________________________________________________________     Values represent the mean ± S.E.M. of the number of animals in     parenthesis.     *Indicates significantly different from untreated control at p < 0.05.     **Indicates significantly different from the control vehicle treated grou     at p < 0.05.

                  TABLE 8    ______________________________________    EFFECT OF LONG TERM APPLICATION (6 WEEKS)    OF LINOLEIC FURAN ON SKIN PROTEIN AND    COLLAGEN SYNTHESIS OF HAIRLESS MICE                 TOTAL PROTEIN                             COLLAGEN                 SYNTHESIS   SYNTHESIS    TREATMENT GROUP                   (DPM  .sup.3 H!-PRO/Punch)    ______________________________________    Control        4,750 ± 630                                 2,830 ± 380    (no treatment) (10)          (10)    Control Vehicle                   5,900 ± 500                                 3,210 ± 290                    (9)           (9)    Vehicle +      10,100 ± 1360*,**                                 4,490 ± 620*    3% natural     (10)          (10)    Linoleic Furan    ______________________________________     Values represent the mean ± S.E.M. of the number of animals of     parenthesis.     *Indicates significantly different from untreated control at p < 0.05.     **Indicates significantly different from the control vehicle treated grou     at p < 0.05.

                  TABLE 9    ______________________________________    EFFECT OF LONG TERM APPLICATION    (6 WEEKS) OF LINOLEIC FURAN ON    SKIN COMPOSITION OF HAIRLESS MICE    TREAT-   EPIDERMAL   DERMAL      TOTAL    MENT     THICKNESS   THICKNESS   THICKNESS    GROUP    (μm)    ______________________________________    Control  2.10 ± 0.4                         18.1 ± 1.1                                     32.7 ± 1.4    (no treat-             (10)        (10)        (10)    ment)    Control  5.86 ± 0.24                         19.4 ± 1.1                                     36.6 ± 1.5    Vehicle  (10)        (10)        (10)    Vehicle +             8.73 ± 0.69*,**                         24.7 ± 0.8*,**                                     43.6 ± 0.9*,**    natural  (10)        (10)        (10)    Linoleic    Furan    ______________________________________     Values represent the mean ± S.E.M. of the number of animals in     parenthesis.     *Indicates significantly different from untreated control at p < 0.05.     **Indicates significantly different from the control vehicle treated grou     at p < 0.05.

                  TABLE 10    ______________________________________    EFFECT OF LONG TERM APPLICATION (6 WEEKS)    OF LINOLEIC FURAN ON THE DERMAL    FIBROBLAST POPULATION OF HAIRLESS MICE    TREAT-  FIBROBLAST POPULATION    MENT    (number of fibroblasts/                            (number of fibroblasts/    GROUP   0.274 mm surface)                            mm.sup.2 cross sectional area)    ______________________________________    Control 121 ± 9      1637 ± 128    (no treat-            (3)             (3)    ment)    Control 242 ± 8*     2730 ± 173*    Vehicle (3)             (3)    Vehicle +            592 ± 54*,** 6174 ± 587*,**    3% natural            (3)             (3)    Linoleic    Furan    ______________________________________     Values represent the mean ± S.E.M. of the number of animals in     parentheses.     *Indicates significantly different from untreated control at p < 0.05.     **Indicates significantly different from control vehicle treated group at     p < 0.05.

Glucose Utilization In Skin After Treatment With Linoleic Furan

PURPOSE

Although there is an increase in general protein synthesis within skintreated with Linoleic Furan, it is not apparent where the extra energynecessary to support this increased activity is generated. An increasein aerobic glucose metabolism may account for a large portion of thisenergy. The Applicant determined that there is an increase in aerobicglucose metabolism.

ANIMAL TREATMENT PROTOCOL

Three groups containing 5 female hairless mice each were obtained. Onegroup served as an untreated control group. The two remaining groupswere treated with 0.1 ml of either vehicle or vehicle plus LinoleicFuran each day for 4 weeks. The treatment groups were as follows:

Control (untreated

Control vehicle

Vehicle+2% Linoleic Furan

Animals were sacrificed at the end of 4 weeks.

ASSAY PROTOCOL

After sacrifice, the skins were removed from the treated area of theanimal and scraped free from the underlying tissues. A 6 mm diameterpunch biopsy was next taken from the skin and placed in a test tube withml of modified Krebs-Ringer solution (0.01M glucose) which had beenaerated with CO₂ :0₂ 5:95% to yield a pH of 7.3. Ten μCi of U-¹⁴C!glucose (ICN Biomedicals, Inc. 6.8 mCi/mmole) were added and the tubesealed and incubated for 45 minutes at 37° C. The reaction was stoppedby the addition of 0.55 ml of 100% (w/v) trichloroacetic acid. The testtubes were incubated for an additional hour, and the released ¹⁴ CO₂ wastrapped on base impregnated paper and subsequently counted in a liquidscintillation counter.

RESULTS

Applicant found that treatment with Linoleic Furan significantlyincreased glucose metabolism (Table 11), and demonstrates that skintreated with Linoleic Furan has enhanced metabolic activity. As olderskin is thought to be less efficient in glucose utilization than youngerskin, the treatment with Linoleic Furan will be useful in treating agedskin.

                  TABLE 11    ______________________________________    EFFECT OF TREATMENT WITH 2% LINOLEIC FURAN    ON SKIN GLUCOSE UTILIZATION OR METABOLISM                   GLUCOSE METABOLISM    TREATMENT      (DPM .sup.14 CO.sub.2 formed/    GROUP          punch/45 min)    ______________________________________    Control        1429 ± 628    (untreated)    (4)    Control vehicle                   1018 ± 182                   (5)    Vehicle +      4042 ± 988**    2% Linoleic Furan    ______________________________________     Values represent the mean ± S.E.M. of the number of animals in     parentheses.     *Indicates significantly different from untreated control at p < 0.05.     **Indicates significantly different from the control vehicle treated grou     at p < 0.05.

The Effect Of Increasing Doses Of Linoleic Furan On Skin Composition AndProtein Synthesis

EXPERIMENTAL PROTOCOL

Five sets of female hairless mice (10 mice per group) were eithertreated daily with 100 μl of a series of cosmetic formulations or usedas untreated control animals. The treatment groups were as follows:

Untreated Control

Control vehicle

Vehicle+1% (w/w) Natural Linoleic Furan

Vehicle+2% (w/w) Natural Linoleic Furan

Vehicle+3% (w/w) Natural Linoleic Furan

The mice were treated for 15 days, sacrificed, and the skin taken forbiochemical analysis, protein synthesis rate determination, andhistology.

RESULTS

From these dose response studies, it was apparent that Linoleic Furanhas a specific effect on skin thickness as determined in a variety ofways (skin thickness, punch biopsy weight, or punch biopsy composition).In addition, the DNA content (a measure of cellularity) was increased ineach vehicle tested when compared to the vehicle control group (Tables12, 13, and 14). These studies also indicated that there was an increasein the protein content due to the delivery of Linoleic Furan. Finally,in every treatment group there was a trend of increased total collagencontent when compared to the control vehicle treated group (Table 12).

In addition to these findings, it was apparent that protein synthesis aswell as collagen synthesis was increased by the addition of LinoleicFuran to the vehicle (Table 13). In addition, the biochemical findingswere strongly supported by the finding that the skin thickness wasincreased in each of the test systems (both epidermis as well as dermis(Table 14)).

                                      TABLE 12    __________________________________________________________________________    EFFECT OF INCREASING DOSES OF    LINOLEIC FURAN ON SKIN COMPOSITION              PUNCH   PROTEIN DNA     COLLAGEN    TREATMENT WEIGHT  CONTENT CONTENT CONTENT    GROUP     (mg/punch)                      (mg/punch)                              (μg/punch)                                      (nmole HYP/punch)    __________________________________________________________________________    Control   11.9 ± 0.4                      1.26 ± 0.08                              67.9 ± 4.6                                      106 ± 12    (no treatment)              (10)    (10)    (10)    (10)    Control vehicle              13.9 ± 0.4                      1.83 ± 0.12*                              55.6 ± 3.1*                                      146 ± 10*              (10)    (10)    (10)    (10)    Vehicle + 14.6 ± 0.7*                      2.08 ± 0.15*                              69.2 ± 4.7**                                      160 ± 14*    1% Linoleic Furan              (10)    (10)    (10)    (10)    Vehicle + 16.1 ± 0.6*,**                      2.07 ± 0.12*                              74.7 ± 4.8**                                      184 ± 25*    2% Linoleic Furan              (10)    (10)    (10)    (10)    Vehicle + 17.8 ± 0.6*,**                      2.51 ± 0.51*,**                              84.2 ± 4.1*,**                                      173 ± 12*    3% Linoleic Furan              (10)     (9)     (9)     (9)    __________________________________________________________________________     Values represent the mean ± S.E.M. of the number of animals in     parentheses.     *Indicates significantly different from untreated control at p > 0.05.     **Indicates significantly different from the control vehicle treated grou     at p < 0.05.

                  TABLE 13    ______________________________________    EFFECT OF INCREASING DOSES OF LINOLEIC FURAN    ON SKIN PROTEIN AND COLLAGEN SYNTHESIS                TOTAL PROTEIN COLLAGEN    TREATMENT   SYNTHESIS     SYNTHESIS    GROUP       (DPM .sup.3 H-PRO/Punch)    ______________________________________    Control     1582 ± 75  960 ± 35    (no treatment)                 (9)           (9)    Control Vehicle                3684 ± 239*                              1350 ± 90*                (10)          (10)    Vehicle ± 1%                3974 ± 462*                              1485 ± 157*    Linoleic Furan                (10)          (10)    Vehicle ± 2%                4623 ± 556*                              1780 ± 232*,**    Linoleic Furan                (10)          (10)    Vehicle ± 3%                5402 ± 724*,**                              1771 ± 248*,**    Linoleic Furan                (10)          (10)    ______________________________________     Values represent the mean ± S.E.M. of the number of animals in     parentheses.     *Indicates significantly different from untreated control at p < 0.05.     **Indicates significantly different from the control vehicle treated grou     at p < 0.05.

                                      TABLE 14    __________________________________________________________________________    EFFECT OF INCREASING DOSES OF    LINOLEIC FURAN ON SKIN MORPHOMETRY                                  TOTAL    TREAT-    EPIDERMAL  DERMAL   THICK-    MENT      THICKNESS  THICKNESS                                  NESS    GROUP     (μm)    (μm)  (μm)    __________________________________________________________________________    Control   2.06 ± 0.03                         16.98 ± 1.03                                  39.74 ± 1.19    (no treatment)              (10)       (10)     (10)    Control Vehicle              4.75 ± 0.15*                         15.09 ± 1.10                                  39.15 ± 1.45              (10)       (10)     (10)    Vehicle + 5.89 ± 0.35*,**                         17.76 ± 0.89                                  40.41 ± 1.60    1% Linoleic Furan              (10)       (10)     (10)    Vehicle + 7.35 ± 0.6*,**                         20.22 ± 1.08*,**                                  41.31 ± 1.65    2% Linoleic Furan              (10)       (10)     (10)    Vehicle + 9.11 ± 0.0.50*,**                         20.74 ± 1.90**                                  42.58 ± 1.55    3% Linoleic Furan              (10)       (10)     (10)    __________________________________________________________________________     Values represent the mean ± S.E.M. of the number of animals in     parentheses.     *Indicates significantly different from untreated control at p < 0.05.     **Indicates significantly different from the control vehicle treated grou     at p < 0.05.

The Combination of Linoleic Furan With Retinol Palmitate

The Lipidic Furans noted above, when administered in combination withretinol palmitate, will have enhanced beneficial therapeutic effectsover and above those of noted with the use of lipidic furans, or thecontrol vehicle alone.

The combination of Linoleic Furan with Retinol Palmitate may betopically or orally administered. The Linoleic Furan may be in eitheroil-continuous or water-continuous phases. The following exampleillustrates one such topical solution:

EXAMPLE 1

    ______________________________________                           Percent    ______________________________________    Water Phase    Water                    60.0-90.0    (Deionized, demineralized or purified)    A C.sub.10 -C.sub.30 alkyl acrylate                             0.1-0.5    cross polymer (an emulsifier)    Preservative solution    A polyoxyethelyne ether  POE!                             0.1-2.0    (e.g. Laureth 4, Oleth 2) or POE Sorbitan ester    (e.g. Polysorbate 20-85)    Oil Phase    Avocado oil (Crude or refined)                              0.0-25.0    Linoleic Furan           2.0    Retinol palmitate        0.1-0.5    An ethylene or propylene glycol ester                             1.0-5.0    (e.g. Glycol stearate)    ______________________________________

In Table 15, the effects of various combinations of control vehicle,oil/water emulsion, retinol palmitate and linoleic furan on the skin ofhairless mice are compared.

                                      TABLE 15    __________________________________________________________________________    EFFECT OF LINOLEIC FURAN WITH RETINOL PALMITATE    ON SKIN OF COMPOSITION OF HAIRLESS MICE                 PUNCH   PROTEIN  DNA      COLLAGEN    TREATMENT    WEIGHT  CONTENT  CONTENT  CONTENT    GROUP        (mg/punch)                         (mg/punch)                                  (mg/punch)                                           (nmole HYP/punch)    __________________________________________________________________________    Control      7.2 ± 1.12                         2.10 ± 0.26                                  4.0 ± 1.0                                           169 ± 24    Oil/Water Emulsion                 10.8 ± 1.15                         2.53 ± 0.17                                  39.2 ± 6.0*,♦                                           174 ± 25    Emulsion    Oil/Water Emulsion +                 11.8 ± 0.12*                         2.64 ± 0.15                                  25.9 ± 3.4*                                           159 ± 22    0.25% Retinol Palmitate    Oil/Water Emulsion +                 16.5 ± 0.72*,**                         3.49 ± 0.18*,**                                  40.2 ± 3.1*,**                                           229 ± 25    0.25% Retinol Palmitate +    2% Linoleic Furan    Oil/Water Emulsion +                 13.9 ± 1.34*,**                         3.16 ± 0.13*,**,♦                                  60.4 ± 2.4*,**,♦                                           183 ± 23    2% Linoleic Furan    __________________________________________________________________________     Values represent the mean ± S.E.M. of five animals in each group.     *Indicates significantly different from untreated control at p ≦     0.05.     **Indicates significantly different from the appropriate vehicle minus     Linoleic Furan treated group at p ≦ 0.5.     ♦Indicates significantly different from the Oil/Water     emulsion + 0.25% Retinol Palmitate treated group at p ≦ 0.05.

Glucose Utilization In Skin After Treatment With Linoleic Furan AndLinoleic Furan With Retinol Palmitate

PURPOSE

To determine if treatment with 2% Linoleic Furan increased glucosemetabolism. Although there is an increase in general protein synthesiswithin skin treated with Linoleic Furan, it is not apparent where theextra energy necessary to support this increased activity is generated.An increase in aerobic glucose metabolism may account for a largeportion of this energy.

ANIMAL TREATMENT PROTOCOL

Four groups containing 5 female mice each were obtained. One groupserved as an untreated control group. The three remaining groups weretreated with 0.1 ml of one of the test substances each day for fourweeks. The treatment groups were as follows:

1) Control (untreated)

2) Vehicle control (Oil in Water Emulsion)

3) Oil in Water Emulsion+2% Linoleic Furan

4) Oil in Water Emulsion+2% Linoleic+0.25% Retinol Palmitate

Animals were sacrificed at the end of four weeks.

ASSAY PROTOCOL

After sacrifice, the skins were removed from the treated area of theanimal and scrapped free from the underlying tissues. A 6 mm diameterpunch biopsy was next taken from the skin and placed in a test tube with5 ml of modified Krebs-Ringer solution (0.01 M glucose) which had beenaerated with CO₂ :0₂ 5:95% to yield a pH of 7.3. Ten μCi of ¹⁴C-U-glucose (ICN Biomedicals, Inc. 6.8 mCl/mmole) were added and thetube sealed and incubated for 45 minutes at 37° C. The reaction wasstopped by the addition of 0.55 ml of 100% (w/v) trichloroacetic acid.The test tubes were incubated for an additional hour and the released ¹⁴CO₂ was trapped on base impregnated paper and subsequently counted in aliquid scintillation counter, yielding the results set forth in Table16.

                  TABLE 16    ______________________________________    EFFECT OF TREATMENT WITH 2% LINOLEIC FURAN    AND RETINOL PALMITATE ON    SKIN GLUCOSE UTILIZATION OR METABOLISM                    GLUCOSE METABOLISM                    (DPM .sup.14 CO.sub.2 formed    TREATMENT       from U-14C-glucose)    GROUP           punch/45 min    ______________________________________    Control         1429 ± 628    (untreated)     (4)    Oil/Water emulsion                    1018 ± 182                    (5)    Oil/Water emulsion +                    4042 ± 988**    2% Linoleic Furan                    (5)    Oil/Water emulsion +                    5007 ± 1122*,**    2% Linoleic Furan +                    (5)    0.25% Retinol Palmitate    ______________________________________     Values indicate the mean ± S.E.M. of the number of animals in     parenthesis.     *Indicates significantly different from untreated control at p ≦     0.05.     **Indicates significantly different from the control vehicle treated grou     at p ≦ 0.05.

DISCUSSION

This study indicates that the treatment with Linoleic Furan and retinolpalmitate significantly increases glucose metabolism. This studydemonstrates that skin treated with Linoleic Furan in combination withretinol palmitate is more "metabolically" active. If, indeed older skinis less efficient in glucose utilization than younger skin, then thiscould be an additional positive statement about the effect of treatmentwith the combination of Linoleic Furan and retinol palmitate.

I claim:
 1. A method for enhancing the metabolism of skin and mucosaltissue and thereby improving its health and appearance, comprising thestep of topically administering to a person's skin in an appropriatecarrier vehicle a lipidic furaris at a concentration range ofapproximately 3%. in combination with retinol palmirate at aconcentration range of approximately 0.1 to 0.5%, said lipidic furanshaving the following general formula: ##STR7## wherein R is selectedfrom the group consisting of one of a straight C₉₋₃₆ alkyl chain, havingthe formula --(C₂)_(n) CH₃, wherein n=8 to 35; a straight unsaturatedalkenyl C₉₋₃₆ chain with a single double bond, of the formula--(CH₂)_(n) --CH═CH--(CH₂)_(m) --CH₃, wherein n and m=0 and n+m=7 to 34,a straight saturated C₉₋₃₆ chain, containing two to six double bonds,with the formula CH₃ --(CH₂)_(m) --(CH═CH)_(x) --(CH₂)_(n) --, whereinm, x and n=0, and m+2x+n=8 to 35; a straight unsaturated chain, havingthe formula --(CH₂)₇ CH═CH--CH₂ --CH═CH(CH₂)₆ --CH₃, a straightunsaturated C₉₋₃₆ chain containing one to six double bonds and onetriple bond, having the formula CH₃ --(CH₂)_(m)--C.tbd.CH--(CH═CH--CH₂)_(x) --(CH₂)_(n) --, wherein m, x and n=0 andm+3x+n=6 to 33, a straight unsaturated chain C₉₋₃₆ containing one to sixdouble bonds and two to six triple bonds, having the structure CH₃--(CH₂)_(m) --(C.tbd.C--CH₂)_(y) --(CH═C--CH₂)_(x) --(CH₂)_(n) --,wherein m, y, x and n=o and m+3y+3x+n=8 to
 35. 2. The method of claim 1,wherein R is --(CH₂)₇ CH═CH--CH₂ --CH═CH(CH₂)₄ --CH₃, and all doublebonds are cis.
 3. A method for enhancing the metabolism of skin andmucosai tissue and thereby improving its health and appearance,comprising the step of administering by topical application to aperson's skin in an appropriate carrier vehicle a lipidic furan at aconcentration range of approximately 1 to 33, in combination withretinol palmitate at a concentration range of about 0.1 to 0.5%,, saidlipidic furan having the following formula: ##STR8##
 4. A method forenhancing the metabolism of skin and mucosal tissue and therebyimproving its health and appearance, comprising the step of topicallyadministering to a person's skin in an appropriate carrier vehiclelinoleic furan at a concentration range of approximately 3% incombination with retinol palmitate at a. concentration range ofapproximately 0.1 to 0.5%.